12042014- I see week 11&12, but I don't see any information pertaining to it, and you are also missing week 13
Weeks 11 and 12
1162014- Good job Weeks 8, 9 and 10
Analysis (Trial 2): PCR Squared was successful this time around, but after preparing the master mix I began to dispense 50 uL of the mix into separate PCR tubes, the first three had the same amount and the fourth PCR tube had less than 20 uL of the mix. Once more, I had already added the Q5 polymerase so I didn't have much time to figure out what went wrong and I just placed the four PCR tubes in the machine. When I ran the gel, I used 13 uL of the PCR Squared sample from the three PCR tubes that looked fine and only 6 from the 4th PCR that did not have enough sample. Lanes 5-7 contained a band at the correct spot (around 735 bp) and those were the samples that had enough mix. Lane 8 did not contain a band and that was probably because i didn't place enough sample for it to show up or the PCR failed because there was not enough sample. The mistake of not having enough sample for each tube cannot be my fault because since three of the four were successful, which means I added everything correctly and it is the pipette that was dispensing 50 uL. I am not sure I can move on to PCR cleanup, because even though PCR Squared was successful, there is heavy smearing in the lanes.
(10/30/14) Figure 22. This is the same gel as Figure 21, except the light contrast was turned down to make the bands formed by PCR Squared easier to see.
(10/30/14) Figure 21. UV image of the Gel Electrophoresis done in 1% Agarose Gel in TAE of the CDS after running PCR Squared. The lanes contain a 1kb DNA ladder, Daren's PCR samples and 4 of my samples after running PCR Squared. A virtual ladder is added for comparison. (Successful)
Lane 1: Skip
Lane 2: 1kb DNA ladder.
Lane 3: Daren's Sample
Lane 4: Skip
Lane 5: PCR Squared Sample
Lane 6: PCR Squared Sample
Lane 7: PCR Squared Sample
Lane 8: PCR Squared Sample
Analysis (Trial 1): PCR Squared failed, but the gel ran successfully because I placed both my two Secondary PCR samples, one that successes and one that failed, into lanes 2 and 3 respectively. This is reflected on the gel since Lane 2 contains the band around the 700 bp mark, and the size of the protein is 735 bp, whereas in lane 3, where my secondary PCR sample failed, it shows no bands in the gel. There are no bands being shown in lanes 6, 7 and 8, which are my samples after PCR Squared, and it was probably due to an error made preparing the master mix. After preparing the 200 uL mix, I began dispensing 50 uL of the master mix into individual PCR tubes, and by the time I was pipetting the 3rd 50 uL sample, there was no more master mix. I either skipped some material when preparing the master mix and thus causing the PCR Squared to fail, or the pipette dispensed the wrong amount into the first two PCR tubes but since I had already added to polymerase, I had to insert the PCR tubes into the PCR machine with unknown amount of master mix within each one which ultimately lead to having no bands showing up in the gel. I need to run PCR Squared and add the appropriate amount of mix into each tube.
Figure 20. UV image of the Gel Electrophoresis done in 1% Agarose Gel in TAE of the CDS after running PCR Squared. The lanes contain a 1kb DNA ladder, two secondary PCR samples and 3 of my samples after running PCR Squared. A virtual ladder is added for comparison. (Failed)
Lane 1: 1kb DNA ladder.
Lane 2: Secondary PCR Sample (2nd Trial)
Lane 3: Secondary PCR Sample (1st Trial)
Lane 4: Skip
Lane 5: 1kb DNA ladder
Lane 6: PCR Squared Sample
Lane 7: PCR Squared Sample
Lane 8: PCR Squared Sample
PCR Squared (10-29-14) First Trial
Analysis (Trial 3): Secondary PCR finally worked. The only change I made was cut down the number of cycles that the PCR runs through from 30 to 25. The band in Lane 5 corresponds to my secondary PCR sample, and it is located near the 750 marker, which is a good result because the size of my target is 735 bp. The next step is to run PCR Squared and amplify the amount of DNA.
Figure 19. UV image of the Gel Electrophoresis done in 1% Agarose Gel in TAE of the CDS after running the secondary PCR. The lanes contain a 1kb DNA ladder, Daren's primary PCR and my own sample after running secondary PCR. (Successful)
Lane 1: Skip.
Lane 2: 1kb DNA ladder.
Lane 3: Daren's Sample (Failed)
Lane 4: Skip
Lane 5: My STP1 sample after running it through Primary and Secondary PCR
Secondary PCR (10-17-14) Third Trial
10232014- Great Job
Weeks 5, 6 and 7
Analysis (Trial 2): The second trail at running the same primers created from Secondary PCR through the gel failed as well. The difference now being that Saniya got a band around 750 bp, which is near the size of our target, which means that her PCR worked as well as the gel. I will run Secondary PCR again and change a couple of things, in the PCR, in order to get desired results.
(10/10/14) Figure 18. UV image of the Gel Electrophoresis done in 1% Agarose Gel in TAE of the CDS after running the secondary PCR. The lanes contain a 10kb DNA ladder, Stacy's Secondary PCR, Saniya's Secondary PCR and my own sample after secondary PCR. (FAILED)
Lane 1: Skip.
Lane 2: 10kb DNA ladder.
Lane 3: Stacy's Secondary PCR.
Lane 4: Saniya's Secondary PCR.
Lane 5: My STP1 sample after running it through Primary and Secondary PCR
Analysis (Trail 1): The Secondary PCR is supposed to take care of mispriming as it add tail end primers into the oligo mix, and therefore creating a CDS of our target with the correct order and size of oligos within its sequence. After running the gel on the sample, there should be one distinct band at the size of the target, but instead there is nothing. The reason being is that the lanes didn't run correctly in the gel because the 100b ladder showed up as a smear instead of the expected ladder. Another gel will need to be ran in order to identify if secondary PCR worked or not.
Figure 17. Inverted UV image of the Gel Electrophoresis done in 1% Agarose Gel in TAE of the CDS after running the secondary PCR. The lanes contain 1kb DNA ladder, 100b DNA ladder, Brianna's Secondary PCR and my own sample after secondary PCR. (FAILED)
Lane 1: Skip.
Lane 2: 1kb DNA ladder.
Lane 3: 100b DNA ladder.
Lane 4: Brianna's Secondary PCR.
Lane 5: STP1 sample after being run through Primary and Secondary PCR.
Secondary PCR (10-08-14)
Analysis: The oligos ordered were connected through Primary PCR, but since there was some misprimes, not all the primers were the right size and therefore it shows the band of smear being more darker, meaning a higher concentration, of the connected near the actual size of our target. The results showed that the PCR worked and we are ready for secondary PCR.
Figure 16. UV image of the Gel Electrophoresis done in 1% Agarose Gel in TAE of the CDS after running the primary PCR. The first 6 lanes are someone else's samples, whereas my sample is within the labeled lane 1 and 2.
Lane 1: 1kb DNA Ladder.
Lane 2: CDS of STP1 after running primary PCR.
Primary PCR (9-30-14)
Analysis: Midi-prep is run to isolate the recombinant pNIC-Bsa4 DNA plasmids from everything else within the pellets that were created after transforming them from BL21-DH5 alpha cells. The concentration acquired was 68.45 ng/uL, which is a decent concentration of plasmids when compared to the desired concentration of 100 ug/uL.
Figure 15. The Nanodrop reading of pNIC-Bsa4 recombinant DNA, that contains the maximum absorbance of 0.550 at the 260nm wavelength and the concentration of 68.6 ng/uL.
Figure 14. The Nanodrop reading of pNIC-Bsa4 recombinant DNA, that has the maximum absorbance of 0.514 at the 260nm wavelength and the concentration of 68.3 ng/uL.
Plasmid Midi-Prep (10-01-14)
Analysis: In order to successfully run PCR to amplify the CDS, Coding DNA Sequence, of our target, Oligos were synthesized and ordered. The CDS was acquired through the NCIB website, and from there the primers were designed using the DNAWorks website, and the tail primers are needed to synthesize compatible ends and prevent misprimes. Once PCR Squared, which amplifies the amount of our target, is successful, the CDS can be inserted into the pNIC-Bsa4 plasmid in order to create a recombinant DNA that can transform the BL21-alpha E. Coli cells and be used for cloning.
Figure 13. The location of where the BsaI enzyme will cut the pNIC-Bsa4 plasmid in order to allow our STP1 gene to be inserted into it.
Figure 12. The sequences of the forward and reverse primers of STP1.
Primer Tail Design (09-29-14)
09242014- Fantastic work!
Weeks 3-4
Analysis: The DNA Ladder in lane one is the incorrect one for PCR, instead of using 1kb, I should have used the 100bp marker. The expected results were that the bands formed in the Lanes 6-8 should have had one band in each lane near the 500bp marker and with each increasing lane the band should have been darker on the Inverted UV image due to it having increasing concentration of DNA plasmid. Lane 6 is the lightest of the bands, followed by Lane 7, but instead of Lane 8 having the darkest band, it does not contain any bands in its lane. However, Lane 9, which is expected to not contain any bands due to it not containing any DNA plasmid, actually has the darkest band. The error that most likely caused by me adding the Sample C to Lane 9 and Sample D to Lane 8. If that is the case, then the results are what were expected, because Sample A has a concentration ratio of 1:10,000, thus leaving the lightest band when compared to the other two samples. Sample B has a concentration ratio of 1:1,000, therefore it has a darker band than Sample A but lighter than Sample C. In Lane 9, Sample C is there with a plasmid concentration ratio of 1:100 and it has the darkest band and finally Sample D, that contains no DNA, leaves no bands in Lane 8. The results from Gel Electrophoresis conclude that the purple protein coding were successfully amplified after running PCR, so the next step would be to purify the DNA by running PCR again or use plasmid for RE Digest.
Figure 11. Inverted UV image of the Gel Electrophoresis of pGBR22 done in 1% Agarose Gel in TAE. The lanes contain a 1kb DNA Ladder, uncut plasmid sample, pGBR22 with restriction enzymes and PCR samples.
Lane 1: 1kb DNA Ladder.
Lane 2: Uncut pGBR22 plasmid with the concentration of 9.729 ng/uL.
Lane 3: Sample 1- pGBR22 with 10 units of EcoRI (RE Digest).
Lane 4: Sample 2- pGBR22 with 10 units of PvuII (RE Digest).
Lane 5: Sample 3- pGBR22 with 5 units of EcoRi and 5 units of PvuII (RE Digest).
Lane 6: Sample A- pGBR22 (concentration of 0.09729 ng/uL) with 1uL of forward primer, reverse primer and Taq after PCR.
Lane 7: Sample B- pGBR22 (concentration of 0.9729 ng/uL) with 1uL of forward primer, reverse primer and Taq after PCR.
Lane 8: Sample D- pGBR22 (concentration of 9.729 ng/uL) with 1uL of forward primer, reverse primer and Taq after PCR.
Lane 9: No DNA plasmid with 1uL of forward primer, reverse primer and Taq after PCR.
Lane 10: Empty
PCR (09-16-14)
Analysis: The Nanodrop readings, Figures 5 and 6, were used to determine the concentration of the pGBR22 sample in order to calculate how much of the sample to add in the 1.7 mL centrifuge tube to start digestion. The average concentration is and 1.55 uL of plasmids were used for each digestion and were placed in tubes used to fill Lanes 3 - 5. There are no bands in Lane 2, which is expected to have a solid band near the 1000bp mark due to the size of pGBR22, the reason it most likely did not contain a band could have been due to not having enough plasmid in the lane and it just did not show in the gel. Lanes 3-5 were expected to show a band where the Restriction Enzymes cut in their DNA, but instead they show smearing at the end of the gel with no bands forming in their lanes. That was caused either by the plasmids being contaminated or adding the buffer in the wrong conditions and causing the Restriction Enzyme to cut the DNA at various random spots. Lanes 6-9 are being used for PCR and Lane 10 was empty. Since the RE Digest failed in giving results, the next step would be run it again in order verify that the transformed DNA is not contaminated and is a good sample to begin cloning.
Figure 10. Inverted UV image of the Gel Electrophoresis of pGBR22 done in 1% Agarose Gel in TAE. The lanes contain a 1kb DNA Ladder, uncut plasmid sample, pGBR22 with restriction enzymes and PCR products.
Lane 1: 1kb DNA Ladder.
Lane 2: Uncut pGBR22 plasmid with the concentration of 9.729 ng/uL.
Lane 3: Sample 1- pGBR22 with 10 units of EcoRI (RE Digest).
Lane 4: Sample 2- pGBR22 with 10 units of PvuII (RE Digest).
Lane 5: Sample 3- pGBR22 with 5 units of EcoRi and 5 units of PvuII (RE Digest).
Lane 6: Sample A- pGBR22 (concentration of 0.09729 ng/uL) with 1uL of forward primer, reverse primer and Taq after PCR.
Lane 7: Sample B- pGBR22 (concentration of 0.9729 ng/uL) with 1uL of forward primer, reverse primer and Taq after PCR.
Lane 8: Sample D- pGBR22 (concentration of 9.729 ng/uL) with 1uL of forward primer, reverse primer and Taq after PCR.
Lane 9: No DNA plasmid with 1uL of forward primer, reverse primer and Taq after PCR.
Lane 10: Empty
Figure 9. The virtual gel from NEB Cutters of the restriction enzymes EcoRI and PvuII. The left is the ladder for EcoRI, the middle one is for PvuII, and the right one is for both EcoRI and PvuII in the same lane.
Figure 8. Nanodrop reading of a sample of pGBR22, that has a theoretical concentration of 972.9 ug/uL. The sample has a maximum absorbance of 19.283 at the 260 nm wavelength and a concentration of 964.1 ug/uL.
Figure 7. Nanodrop reading of a sample of pGBR22, that has a theoretical concentration of 972.9 ug/uL. The sample has a maximum absorbance of 19.385 at the 260 nm wavelength and a concentration of 969.3 ug/uL.
Restriction Enzyme Digest (09-16-14)
==
==
Weeks 1 & 2
Analysis:This lab was very successful; the pGBR22 sequence was completed. The process began by acquiring a sequence and trimming it down by removing unrecognized base pairs, a mistake in deleting base pairs could cause a huge change in the DNA sequence because it could lead to a mutation in the sequence. The next step is to go to BLAST and use their database to match the M13 sequence to any other sequences to see if they matched, the best match for M13 had a 60% query cover and 100% identity match with ‘Montipora efflorescens GFP-like chromoprotein mRNA, complete cds.’ Next step was to acquire the sequence for the one that matched best and use BLAST again to align their sequences to see if they align and catch any mismatches in the sequences. It is very important to use BLAST nucleotides database because it would be matching it to protein sequences if the BLAST protein database was being used. After matching it, we translated the sequence so make sure there were no errors and matched them up using BLAST proteins to make sure mutations are not an issue for the DNA. After making sure that both the sequences match and that there are no errors, we can insert the M13R sequence into the pGemT sequence to create a full pGBR22 sequence.
Figure 6. The sequence is the complete pGBR22 sequence after the M13Rev sequence was inserted into the pGemT Reverse compliment sequence. The gray highlight signifies the left flanking region and the purple is the right flanking region. The underlined segment is the M13Rev sequence; the start codon is highlighted green, the Shine-Dalgarno sequences are cyan, the stop codon is red and the M13 Reverse primer site is underlined and bold.
Figure 5. The DNA sequence of pGBR22 created by the UT DNA Sequencing Facility. It contains the base pairs of the sequence and the N's are base pairs that could not be determined.
Analyze DNA Sequences (09-05-14)
Analysis: The colonies grown on the plates are the E. coli cells that were transformed using the pNIC-Bsa4 plasmids and became resistant to kanamycin that was in the LB plate. The difference in growth was due to the fact that in Figure 3, 50 uL of pNIC-Bsa4 were used and in Figure 4, only 10uL were used.. There are other factors that could have caused the colony growth since I made the mistake of not having a control plate, so the next step is to express the proteins and purify them in order to confirm the transformation.
Figure 4. Colony growth can be seen in the LB plate that contains kanamycin, SOC media and 10uL of pNIC-Bsa4 plasmids that were transformed into DH5-alpha E. coli cells after being incubated overnight at 37 C.
Figure 3. Colony growth can be seen in the LB plate that contains kanamycin, SOC media and 50uL of pNIC-Bsa4 plasmids that were transformed into DH5-alpha E. coli cells after being incubated overnight at 37 C.
Transformation of DH5-alpha E. coli Cells (pNIC-Bsa4 Plasmids) (09-03-14)
Analysis: The Nanodrop readings below, Figure 1 and 2, were taken to determine the concentration and purity of the sample pGBR22. The average concentration of pGBR22 is 235.1 ng/uL. The 260/280 ratio is 1.90 and the 260/230 ratio is 1.92, that are close to the ratios of 260/280 and 260/230 ratios for pure pGBR22, which are 2.1 and 1.9 respectively. The values from the readings confirm that the pGBR22 sample used is close to being pure and can be used to send to the UT DNA Sequencing Facility to determine the sequence of the sample.
Figure 2. The Nanodrop reading of pGBR22, that gives us the maximum absorbance of 4.264 at the 260nm wavelength and the concentration of 213.2 ng/uL.
Figure 1. The Nanodrop reading of pGBR22, that gives us the maximum absorbance of 5.139 at the 260nm wavelength and the concentration of 257.0 ng/uL.
12042014- I see week 11&12, but I don't see any information pertaining to it, and you are also missing week 13
Weeks 11 and 121162014- Good job
Weeks 8, 9 and 10
Analysis (Trial 2): PCR Squared was successful this time around, but after preparing the master mix I began to dispense 50 uL of the mix into separate PCR tubes, the first three had the same amount and the fourth PCR tube had less than 20 uL of the mix. Once more, I had already added the Q5 polymerase so I didn't have much time to figure out what went wrong and I just placed the four PCR tubes in the machine. When I ran the gel, I used 13 uL of the PCR Squared sample from the three PCR tubes that looked fine and only 6 from the 4th PCR that did not have enough sample. Lanes 5-7 contained a band at the correct spot (around 735 bp) and those were the samples that had enough mix. Lane 8 did not contain a band and that was probably because i didn't place enough sample for it to show up or the PCR failed because there was not enough sample. The mistake of not having enough sample for each tube cannot be my fault because since three of the four were successful, which means I added everything correctly and it is the pipette that was dispensing 50 uL. I am not sure I can move on to PCR cleanup, because even though PCR Squared was successful, there is heavy smearing in the lanes.
Figure 22. This is the same gel as Figure 21, except the light contrast was turned down to make the bands formed by PCR Squared easier to see.
Figure 21. UV image of the Gel Electrophoresis done in 1% Agarose Gel in TAE of the CDS after running PCR Squared. The lanes contain a 1kb DNA ladder, Daren's PCR samples and 4 of my samples after running PCR Squared. A virtual ladder is added for comparison. (Successful)
Lane 1: Skip
Lane 2: 1kb DNA ladder.
Lane 3: Daren's Sample
Lane 4: Skip
Lane 5: PCR Squared Sample
Lane 6: PCR Squared Sample
Lane 7: PCR Squared Sample
Lane 8: PCR Squared Sample
Analysis (Trial 1): PCR Squared failed, but the gel ran successfully because I placed both my two Secondary PCR samples, one that successes and one that failed, into lanes 2 and 3 respectively. This is reflected on the gel since Lane 2 contains the band around the 700 bp mark, and the size of the protein is 735 bp, whereas in lane 3, where my secondary PCR sample failed, it shows no bands in the gel. There are no bands being shown in lanes 6, 7 and 8, which are my samples after PCR Squared, and it was probably due to an error made preparing the master mix. After preparing the 200 uL mix, I began dispensing 50 uL of the master mix into individual PCR tubes, and by the time I was pipetting the 3rd 50 uL sample, there was no more master mix. I either skipped some material when preparing the master mix and thus causing the PCR Squared to fail, or the pipette dispensed the wrong amount into the first two PCR tubes but since I had already added to polymerase, I had to insert the PCR tubes into the PCR machine with unknown amount of master mix within each one which ultimately lead to having no bands showing up in the gel. I need to run PCR Squared and add the appropriate amount of mix into each tube.
Figure 20. UV image of the Gel Electrophoresis done in 1% Agarose Gel in TAE of the CDS after running PCR Squared. The lanes contain a 1kb DNA ladder, two secondary PCR samples and 3 of my samples after running PCR Squared. A virtual ladder is added for comparison. (Failed)
Lane 1: 1kb DNA ladder.
Lane 2: Secondary PCR Sample (2nd Trial)
Lane 3: Secondary PCR Sample (1st Trial)
Lane 4: Skip
Lane 5: 1kb DNA ladder
Lane 6: PCR Squared Sample
Lane 7: PCR Squared Sample
Lane 8: PCR Squared Sample
PCR Squared (10-29-14) First Trial
Analysis (Trial 3): Secondary PCR finally worked. The only change I made was cut down the number of cycles that the PCR runs through from 30 to 25. The band in Lane 5 corresponds to my secondary PCR sample, and it is located near the 750 marker, which is a good result because the size of my target is 735 bp. The next step is to run PCR Squared and amplify the amount of DNA.
Figure 19. UV image of the Gel Electrophoresis done in 1% Agarose Gel in TAE of the CDS after running the secondary PCR. The lanes contain a 1kb DNA ladder, Daren's primary PCR and my own sample after running secondary PCR. (Successful)
Lane 1: Skip.
Lane 2: 1kb DNA ladder.
Lane 3: Daren's Sample (Failed)
Lane 4: Skip
Lane 5: My STP1 sample after running it through Primary and Secondary PCR
Secondary PCR (10-17-14) Third Trial
10232014- Great Job
Weeks 5, 6 and 7
Analysis (Trial 2): The second trail at running the same primers created from Secondary PCR through the gel failed as well. The difference now being that Saniya got a band around 750 bp, which is near the size of our target, which means that her PCR worked as well as the gel. I will run Secondary PCR again and change a couple of things, in the PCR, in order to get desired results.
Figure 18. UV image of the Gel Electrophoresis done in 1% Agarose Gel in TAE of the CDS after running the secondary PCR. The lanes contain a 10kb DNA ladder, Stacy's Secondary PCR, Saniya's Secondary PCR and my own sample after secondary PCR. (FAILED)
Lane 1: Skip.
Lane 2: 10kb DNA ladder.
Lane 3: Stacy's Secondary PCR.
Lane 4: Saniya's Secondary PCR.
Lane 5: My STP1 sample after running it through Primary and Secondary PCR
Analysis (Trail 1): The Secondary PCR is supposed to take care of mispriming as it add tail end primers into the oligo mix, and therefore creating a CDS of our target with the correct order and size of oligos within its sequence. After running the gel on the sample, there should be one distinct band at the size of the target, but instead there is nothing. The reason being is that the lanes didn't run correctly in the gel because the 100b ladder showed up as a smear instead of the expected ladder. Another gel will need to be ran in order to identify if secondary PCR worked or not.
Figure 17. Inverted UV image of the Gel Electrophoresis done in 1% Agarose Gel in TAE of the CDS after running the secondary PCR. The lanes contain 1kb DNA ladder, 100b DNA ladder, Brianna's Secondary PCR and my own sample after secondary PCR. (FAILED)
Lane 1: Skip.
Lane 2: 1kb DNA ladder.
Lane 3: 100b DNA ladder.
Lane 4: Brianna's Secondary PCR.
Lane 5: STP1 sample after being run through Primary and Secondary PCR.
Secondary PCR (10-08-14)
Analysis: The oligos ordered were connected through Primary PCR, but since there was some misprimes, not all the primers were the right size and therefore it shows the band of smear being more darker, meaning a higher concentration, of the connected near the actual size of our target. The results showed that the PCR worked and we are ready for secondary PCR.
Figure 16. UV image of the Gel Electrophoresis done in 1% Agarose Gel in TAE of the CDS after running the primary PCR. The first 6 lanes are someone else's samples, whereas my sample is within the labeled lane 1 and 2.
Lane 1: 1kb DNA Ladder.
Lane 2: CDS of STP1 after running primary PCR.
Primary PCR (9-30-14)
Analysis: Midi-prep is run to isolate the recombinant pNIC-Bsa4 DNA plasmids from everything else within the pellets that were created after transforming them from BL21-DH5 alpha cells. The concentration acquired was 68.45 ng/uL, which is a decent concentration of plasmids when compared to the desired concentration of 100 ug/uL.
Figure 15. The Nanodrop reading of pNIC-Bsa4 recombinant DNA, that contains the maximum absorbance of 0.550 at the 260nm wavelength and the concentration of 68.6 ng/uL.
Figure 14. The Nanodrop reading of pNIC-Bsa4 recombinant DNA, that has the maximum absorbance of 0.514 at the 260nm wavelength and the concentration of 68.3 ng/uL.
Plasmid Midi-Prep (10-01-14)
Analysis: In order to successfully run PCR to amplify the CDS, Coding DNA Sequence, of our target, Oligos were synthesized and ordered. The CDS was acquired through the NCIB website, and from there the primers were designed using the DNAWorks website, and the tail primers are needed to synthesize compatible ends and prevent misprimes. Once PCR Squared, which amplifies the amount of our target, is successful, the CDS can be inserted into the pNIC-Bsa4 plasmid in order to create a recombinant DNA that can transform the BL21-alpha E. Coli cells and be used for cloning.
Figure 13. The location of where the BsaI enzyme will cut the pNIC-Bsa4 plasmid in order to allow our STP1 gene to be inserted into it.
Figure 12. The sequences of the forward and reverse primers of STP1.
Primer Tail Design (09-29-14)
09242014- Fantastic work!
Weeks 3-4
Analysis: The DNA Ladder in lane one is the incorrect one for PCR, instead of using 1kb, I should have used the 100bp marker. The expected results were that the bands formed in the Lanes 6-8 should have had one band in each lane near the 500bp marker and with each increasing lane the band should have been darker on the Inverted UV image due to it having increasing concentration of DNA plasmid. Lane 6 is the lightest of the bands, followed by Lane 7, but instead of Lane 8 having the darkest band, it does not contain any bands in its lane. However, Lane 9, which is expected to not contain any bands due to it not containing any DNA plasmid, actually has the darkest band. The error that most likely caused by me adding the Sample C to Lane 9 and Sample D to Lane 8. If that is the case, then the results are what were expected, because Sample A has a concentration ratio of 1:10,000, thus leaving the lightest band when compared to the other two samples. Sample B has a concentration ratio of 1:1,000, therefore it has a darker band than Sample A but lighter than Sample C. In Lane 9, Sample C is there with a plasmid concentration ratio of 1:100 and it has the darkest band and finally Sample D, that contains no DNA, leaves no bands in Lane 8. The results from Gel Electrophoresis conclude that the purple protein coding were successfully amplified after running PCR, so the next step would be to purify the DNA by running PCR again or use plasmid for RE Digest.
Figure 11. Inverted UV image of the Gel Electrophoresis of pGBR22 done in 1% Agarose Gel in TAE. The lanes contain a 1kb DNA Ladder, uncut plasmid sample, pGBR22 with restriction enzymes and PCR samples.
Lane 1: 1kb DNA Ladder.
Lane 2: Uncut pGBR22 plasmid with the concentration of 9.729 ng/uL.
Lane 3: Sample 1- pGBR22 with 10 units of EcoRI (RE Digest).
Lane 4: Sample 2- pGBR22 with 10 units of PvuII (RE Digest).
Lane 5: Sample 3- pGBR22 with 5 units of EcoRi and 5 units of PvuII (RE Digest).
Lane 6: Sample A- pGBR22 (concentration of 0.09729 ng/uL) with 1uL of forward primer, reverse primer and Taq after PCR.
Lane 7: Sample B- pGBR22 (concentration of 0.9729 ng/uL) with 1uL of forward primer, reverse primer and Taq after PCR.
Lane 8: Sample D- pGBR22 (concentration of 9.729 ng/uL) with 1uL of forward primer, reverse primer and Taq after PCR.
Lane 9: No DNA plasmid with 1uL of forward primer, reverse primer and Taq after PCR.
Lane 10: Empty
PCR (09-16-14)
Analysis: The Nanodrop readings, Figures 5 and 6, were used to determine the concentration of the pGBR22 sample in order to calculate how much of the sample to add in the 1.7 mL centrifuge tube to start digestion. The average concentration is and 1.55 uL of plasmids were used for each digestion and were placed in tubes used to fill Lanes 3 - 5. There are no bands in Lane 2, which is expected to have a solid band near the 1000bp mark due to the size of pGBR22, the reason it most likely did not contain a band could have been due to not having enough plasmid in the lane and it just did not show in the gel. Lanes 3-5 were expected to show a band where the Restriction Enzymes cut in their DNA, but instead they show smearing at the end of the gel with no bands forming in their lanes. That was caused either by the plasmids being contaminated or adding the buffer in the wrong conditions and causing the Restriction Enzyme to cut the DNA at various random spots. Lanes 6-9 are being used for PCR and Lane 10 was empty. Since the RE Digest failed in giving results, the next step would be run it again in order verify that the transformed DNA is not contaminated and is a good sample to begin cloning.
Figure 10. Inverted UV image of the Gel Electrophoresis of pGBR22 done in 1% Agarose Gel in TAE. The lanes contain a 1kb DNA Ladder, uncut plasmid sample, pGBR22 with restriction enzymes and PCR products.
Lane 1: 1kb DNA Ladder.
Lane 2: Uncut pGBR22 plasmid with the concentration of 9.729 ng/uL.
Lane 3: Sample 1- pGBR22 with 10 units of EcoRI (RE Digest).
Lane 4: Sample 2- pGBR22 with 10 units of PvuII (RE Digest).
Lane 5: Sample 3- pGBR22 with 5 units of EcoRi and 5 units of PvuII (RE Digest).
Lane 6: Sample A- pGBR22 (concentration of 0.09729 ng/uL) with 1uL of forward primer, reverse primer and Taq after PCR.
Lane 7: Sample B- pGBR22 (concentration of 0.9729 ng/uL) with 1uL of forward primer, reverse primer and Taq after PCR.
Lane 8: Sample D- pGBR22 (concentration of 9.729 ng/uL) with 1uL of forward primer, reverse primer and Taq after PCR.
Lane 9: No DNA plasmid with 1uL of forward primer, reverse primer and Taq after PCR.
Lane 10: Empty
Figure 9. The virtual gel from NEB Cutters of the restriction enzymes EcoRI and PvuII. The left is the ladder for EcoRI, the middle one is for PvuII, and the right one is for both EcoRI and PvuII in the same lane.
Figure 8. Nanodrop reading of a sample of pGBR22, that has a theoretical concentration of 972.9 ug/uL. The sample has a maximum absorbance of 19.283 at the 260 nm wavelength and a concentration of 964.1 ug/uL.
Figure 7. Nanodrop reading of a sample of pGBR22, that has a theoretical concentration of 972.9 ug/uL. The sample has a maximum absorbance of 19.385 at the 260 nm wavelength and a concentration of 969.3 ug/uL.
Restriction Enzyme Digest (09-16-14)
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Weeks 1 & 2
Analysis: This lab was very successful; the pGBR22 sequence was completed. The process began by acquiring a sequence and trimming it down by removing unrecognized base pairs, a mistake in deleting base pairs could cause a huge change in the DNA sequence because it could lead to a mutation in the sequence. The next step is to go to BLAST and use their database to match the M13 sequence to any other sequences to see if they matched, the best match for M13 had a 60% query cover and 100% identity match with ‘Montipora efflorescens GFP-like chromoprotein mRNA, complete cds.’ Next step was to acquire the sequence for the one that matched best and use BLAST again to align their sequences to see if they align and catch any mismatches in the sequences. It is very important to use BLAST nucleotides database because it would be matching it to protein sequences if the BLAST protein database was being used. After matching it, we translated the sequence so make sure there were no errors and matched them up using BLAST proteins to make sure mutations are not an issue for the DNA. After making sure that both the sequences match and that there are no errors, we can insert the M13R sequence into the pGemT sequence to create a full pGBR22 sequence.
Figure 6. The sequence is the complete pGBR22 sequence after the M13Rev sequence was inserted into the pGemT Reverse compliment sequence. The gray highlight signifies the left flanking region and the purple is the right flanking region. The underlined segment is the M13Rev sequence; the start codon is highlighted green, the Shine-Dalgarno sequences are cyan, the stop codon is red and the M13 Reverse primer site is underlined and bold.
Figure 5. The DNA sequence of pGBR22 created by the UT DNA Sequencing Facility. It contains the base pairs of the sequence and the N's are base pairs that could not be determined.
Analyze DNA Sequences (09-05-14)
Analysis: The colonies grown on the plates are the E. coli cells that were transformed using the pNIC-Bsa4 plasmids and became resistant to kanamycin that was in the LB plate. The difference in growth was due to the fact that in Figure 3, 50 uL of pNIC-Bsa4 were used and in Figure 4, only 10uL were used.. There are other factors that could have caused the colony growth since I made the mistake of not having a control plate, so the next step is to express the proteins and purify them in order to confirm the transformation.
Figure 4. Colony growth can be seen in the LB plate that contains kanamycin, SOC media and 10uL of pNIC-Bsa4 plasmids that were transformed into DH5-alpha E. coli cells after being incubated overnight at 37 C.
Figure 3. Colony growth can be seen in the LB plate that contains kanamycin, SOC media and 50uL of pNIC-Bsa4 plasmids that were transformed into DH5-alpha E. coli cells after being incubated overnight at 37 C.
Transformation of DH5-alpha E. coli Cells (pNIC-Bsa4 Plasmids) (09-03-14)
Analysis: The Nanodrop readings below, Figure 1 and 2, were taken to determine the concentration and purity of the sample pGBR22. The average concentration of pGBR22 is 235.1 ng/uL. The 260/280 ratio is 1.90 and the 260/230 ratio is 1.92, that are close to the ratios of 260/280 and 260/230 ratios for pure pGBR22, which are 2.1 and 1.9 respectively. The values from the readings confirm that the pGBR22 sample used is close to being pure and can be used to send to the UT DNA Sequencing Facility to determine the sequence of the sample.
Figure 2. The Nanodrop reading of pGBR22, that gives us the maximum absorbance of 4.264 at the 260nm wavelength and the concentration of 213.2 ng/uL.
Figure 1. The Nanodrop reading of pGBR22, that gives us the maximum absorbance of 5.139 at the 260nm wavelength and the concentration of 257.0 ng/uL.
Nanodrop (08-29-14)